Human Autoreactive CD4+ T Cells from Naive CD45RA + and Memory CD45RO+ Subsets Differ with Respect to Epitope Specificity and Functional Antigen Avidity This information is current as of September 26, 2021. Paolo A. Muraro, Martin Pette, Bibiana Bielekova, Henry F. McFarland and Roland Martin J Immunol 2000; 164:5474-5481; ; doi: 10.4049/jimmunol.164.10.5474 http://www.jimmunol.org/content/164/10/5474 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Human Autoreactive CD4؉ T Cells from Naive CD45RA؉ and Memory CD45RO؉ Subsets Differ with Respect to Epitope Specificity and Functional Antigen Avidity

Paolo A. Muraro,*† Martin Pette,*‡ Bibiana Bielekova,* Henry F. McFarland,* and Roland Martin1*

T cells with specificity for self-Ags are normally present in the peripheral blood, and, upon activation, may target tissue Ags and become involved in the pathogenesis of autoimmune processes. In multiple sclerosis, a demyelinating disease of the CNS, it is postulated that inflammatory damage is initiated by CD4؉ T cells reactive to myelin Ags. To investigate the potential naive vs memory origin of circulating myelin-reactive cells, we have generated myelin basic protein (MBP)- and tetanus toxoid-specific T ؉ ؊ ؉ ؊ ؉ cell clones from CD45RA /RO and CD45RO /RA CD4 T cell subsets from the peripheral blood of multiple sclerosis patients Downloaded from and controls. Our results show that 1) the response to MBP, different from that to TT, predominantly emerges from the CD45RA؉ subset; 2) the reactivity to immunodominant MBP epitopes mostly resides in the CD45RA؉ subset; 3) in each individual, the recognition of single MBP epitopes is skewed to either subset, with no overlap in the Ag fine specificity; and 4) in spite of a lower expression of costimulatory and adhesion molecules, CD45RA؉ subset-derived clones recognize epitopes with higher functional Ag avidity. These findings point to a central role of the naive CD45RA؉ T cell subset as the source for immunodominant, potentially ؉ pathogenic effector CD4 T cell responses in humans. The Journal of Immunology, 2000, 164: 5474–5481. http://www.jimmunol.org/

mmunological research on organ-specific autoimmune dis- (9–11), their TCR signaling (12), and their tissue-specific homing eases has focused to a large extent on the identification of receptor-ligand interactions (13–16). I candidate self-Ags. In this context, myelin proteins have been Since human T cells upon antigenic priming down-regulate the studied as potential targets of autoimmune reactivity in the patho- high-m.w. CD45RA isoform and reciprocally up-regulate the low- genesis of multiple sclerosis (MS)2, the most common demyeli- m.w. form CD45RO, these molecules have been proposed as nating disease of the CNS. Ags such as myelin basic protein markers for naive (CD45RAϩ/ROϪ) and memory (CD45RAϪ/ (MBP) have been employed as immunogens in vivo to learn from ROϩ) T cells (17, 18). The following well-established observa- experimental disease in animals (experimental allergic encephalo- tions corroborate this notion: 1) CD45ROϩ cells are reduced under by guest on September 26, 2021 myelitis, EAE), and in vitro to study the human immune response conditions of limited antigenic exposure, such as in human cord in MS patients (reviewed in Ref. 1). EAE studies have demon- blood or in neonatal mice; 2) CD45ROϩ cells are almost absent strated that inflammatory demyelinating CNS disorders can be me- ϩ in the spleens of mice bred in a germfree environment; 3) diated by CD4 myelin-specific T cells. Extensive data in humans, increased proportions of CD45ROϩ T cells are found in the on the other hand, indicate that myelin-reactive cells are part of the peripheral blood of aging or immunized humans and animals; normal T cell repertoire both in MS patients and healthy subjects and 4) CD4ϩCD45ROϩ T cells predominantly give rise to the (2–5). However, these studies on the frequency of self-reactive T human response to recall Ags such as tetanus toxoid (TT) in lymphocytes did not address whether these cells had been Ag immunized subjects (19). More recent data, however, demon- primed in the body, i.e., whether they derived from a memory or strated that changes in CD45R isoforms are reversible in the rat naive pool. This is a relevant question, since naive and memory T (20, 21). Since evidence of such reversion in humans is so far only cells play different roles in the immune response (6–8) and differ indirect (22), CD45RA vs CD45RO expression has been widely with respect to their Ag presentation and activation requirements accepted and used to identify and select human naive and memory CD4ϩ T cells, respectively. A concomitant expression of both CD45RA and CD45RO has been associated with the phenotype of *Neuroimmunology Branch, National Institute of Neurological Disorders and Stroke, effector T cells, but the relationship of this cell population with that † National Institutes of Health, Bethesda, MD 20892; Department of Oncology and of naive and memory cells is not fully understood (8, 23). Thus, we Neuroscience, Medical School, University “Gabriele D’Annunzio,” Chieti, Italy; and ϩ ‡Neurologische Klinik, Universita¨tsklinikum Carl-Gustav-Carus der Technischen studied human myelin Ag-specific responses from CD4 T cell Universita¨t Dresden, Dresden, Germany populations identified by clear expression of either the CD45RA or Received for publication July 13, 1999. Accepted for publication March 1, 2000. CD45RO isoform postulating they represent resting naive and The costs of publication of this article were defrayed in part by the payment of page memory T cells, and excluded double-positive cells which may charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. either represent a transitional or an effector state. 1 To better understand the origin, the functional characteristics, Address correspondence and reprint requests to Dr. Roland Martin, Neuroimmu- ϩ nology Branch, National Institute of Neurological Disorders and Stroke, National and the specificity of CD4 MBP-specific T cells derived from the Institutes of Health, 10 Center Drive MSC1400, Bethesda, MD 20892-1400. E-mail peripheral blood of MS patients and controls, we asked 1) whether address: [email protected] autoreactive T cells can originate both from the putatively naive 2 Abbreviations used in this paper: MS, multiple sclerosis; EAE, experimental allergic ϩ Ϫ Ϫ ϩ ϩ encephalomyelitis; MBP, myelin basic protein; SAg, superantigen; TCC, T cell clone; (CD45RA /RO ) and memory (CD45RA /RO ) CD4 subsets; TT, tetanus toxoid; CD40L, CD40 ligand. 2) whether effector autoreactive cells raised from these subsets

Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00 The Journal of Immunology 5475 express different T helper phenotypes; and 3) whether the MBP- with autologous irradiated PBMC (3000 rad, 1.5 ϫ 105/well) as APC and reactive T cell repertoires originating from the two subsets differ stimulated with 30 ␮g/ml MBP or 10 ␮g/ml MBP peptide. Wells contain- ing specific proliferating T cells (stimulation index Ͼ2 with a ␦ Ͼ1000) with respect to frequency, epitope recognition, and antigenic cpm were periodically (10–15 days) restimulated with Ag and APC and ex- affinity. panded in medium containing 5 ng/ml of human rIL-2 (generously pro- vided by Prof. Sebald, Biozentrum, Wu¨rzburg, Germany). Clonality of T Materials and Methods cell cultures was assessed by PCR-assisted TCR V␤ gene usage analysis as Subjects described elsewhere (5). Concentrations of the soluble factors IFN-␥, TNF-␣, IL-4, IL-10, and Donors were typed for HLA class II using standardized molecular typing GM-CSF were measured by ELISA using CytoSets (BioSource, Camarillo, methods by the Department of Transfusion Medicine, National Institutes of CA) according to an optimized standard protocol. Aliquots of cell culture Health. To include well-characterized backgrounds in terms of HLA-re- supernatant (50 ␮l) were harvested 48 h after Ag stimulation and stored at stricted T cell response to MBP, we selected patients with haplotypes in- Ϫ80°C until analysis. Supernatants were diluted 1:5 with assay buffer and cluding DRB1*1501, *1502, or *0401. Age, gender, and HLA-DR haplo- used in a total assay volume of 75 ␮l/well. For each TCC tested, we mea- type of the subjects were as follows: MS patients MS1, 31-year-old female, sured cytokine secretion upon the following conditions: no Ag, 10 and 100 DRB1*1501,04*; MS2, 31-year-old female, DRB1*1501,04*; MS3, 24-year- ␮g/ml MBP, 10 and 100 ␮g/ml specific MBP peptide. All measurements old female, DRB1*0403,1501; MS4, 44-year-old female, DRB1*0401,1301; were performed in duplicate wells, and the specificity of proliferation was MS5, 39-year-old-female, DRB1*1501; MS6, 32-year-old-female, ascertained in every instance. DRB1*1502, 0701; and MS7, 38-year-old male, DRB1*0401,1501 and Antigenic peptide specificities of the MBP-reactive TCC were defined healthy donors: HD1, 33-year-old female, DRB1*0401,07*; HD2, 56-year-old by using a panel of 16 overlapping 19-mers in standard proliferation as- female, DRB1*0401,1302; and HD3, 30-year-old male, DRB1*0301,1101. says. Epitopes within the immunodominant region of MBP(111–129) were The study was approved by the Institute Clinical Research Subpanel, and further mapped using a set of 16 peptides truncated at the amino (112 to informed consent was obtained from each patient. 119–129) and carboxyl (111–121 to 128) termini at equimolar concentra- Downloaded from tions (5). The overall functional Ag affinity was estimated by determining Ags the Ag concentration required to induce 50% of the maximum proliferation Human MBP was prepared according to the method of Deibler et al. (24). (EC50). This was accomplished in dose-response proliferation assays cov- Ϫ5 2 ␮ A single batch of MBP of known purity and in vitro immunogenicity was ering Ag concentrations between 10 and 10 g/ml. used throughout the entire study. MBP peptides were synthesized on a The expression of cell surface markers of MBP-specific TCC was as- Beckman 990 peptide synthesizer (Beckman Coulter, Fullerton, CA) using sessed by three-color flow cytometry staining. Taking into account that the expression of several lymphocyte surface molecules including CD45 iso- Merrifield’s solid-phase synthesis methodology according to published se- http://www.jimmunol.org/ quences (5). The superantigens (SAgs) staphylococcal enteroxin A, staphy- forms varies during cell cycle progression (25), we performed FACS stain- lococccal enteroxin B, staphylococcal enteroxin C-1, -2, -3, staphylococcal ing always on resting TCC at the end of each restimulation cycle (day 12), enteroxin D, staphylococcal enteroxin E, and toxic shock syndrome toxin-1 thus avoiding to introduce a bias. To monitor a possible variation of cell were obtained from Toxin Technologies (Sarasota, FL). TT was purchased surface molecules after repeated antigenic contact, analysis included TCC from Pasteur Merieux Connaught (North York, Ontario, Canada). which underwent a number of restimulations ranging from three to nine cycles. For FACS staining, 1 ϫ 105 T cells/well were plated in 96-well ϩ Purification of CD4 T cell subsets plates and washed twice with 200 ␮l cold staining buffer (PBS 1% FCS- 0.1% (w/v) sodium azide). The following mAb combinations were added PBMC were obtained from leukoapheresis buffy coats by density gradient at saturating concentrations: 1) IgG1 FITC, IgG1 PE, IgG1 Cy-Chrome; 2) centrifugation (Organon Teknika, Durham, NC). T cell subsets were iso- CD45RA FITC (clone HI100), CD45RO PE (clone UCHL1), and CD4 lated from 1–2 ϫ 108 freshly obtained PBMC via high-affinity negative ϩ Ϫ ϩ Ϫ Cy-Chrome; 3) CD28 FITC, CD62L PE, and CD4 Cy-Chrome; 4) CD29 by guest on September 26, 2021 selection using human CD4 /45RO or CD4 /45RA subset column kits FITC, CD54 PE, and CD 4 Cy-Chrome; 5) CD25 FITC, CD40 ligand (R&D Systems, Minneapolis, MN), essentially according to the manufac- (CD40L) PE, and CD4 Cy-Chrome; 6) HLA-DR FITC, CD2 PE, and CD4 turer’s protocol. Our improvements included thorough resuspension of the Cy-Chrome; 7) CD11a FITC, CD95 PE, and CD4 Cy-Chrome; 8) CD49d cell pellet by gently passing the cells six times through a 16-gauge needle FITC, CD27 PE, and CD4 Cy-Chrome; 9) CD80 FITC, CD86 PE, and CD4 before loading and keeping a steady cell elution flow by carefully main- Cy-Chrome; 10) CD30 FITC, CD58 PE, and CD4 Cy-Chrome; 11) CD26 taining a 2-ml wash buffer volume in the column reservoir. Cryopreserved FITC, CD44 PE, and CD4 Cy-Chrome; 12) CD134 FITC, CD152(CTLA-4) cells proved unsuitable for the separation procedure due to reduced via- PE, and CD4 Cy-Chrome; and 13) unstained cells. Abs were purchased from bility and an increased tendency for aggregation. Therefore, only data ob- Becton Dickinson (CD2 PE, CD69 PE, and HLA-DR PE), Exalpha (Boston, tained from subset separations of freshly collected PBMC were included in MA; IgG1 FITC, IgG1 PE), Immunotech (Westbrook, ME; CD11a FITC, this study. Composition of the PBMC and purity of the separated T cell CD25 FITC, and CD49d FITC), and PharMingen (San Diego, CA). After 30 subsets were routinely checked by flow cytometry using a FACScan- and min incubation on ice in the dark, cells were washed twice, resuspended in 200 CellQuest software (Becton Dickinson, Mountain View, CA) after staining ␮l staining buffer, and transferred to 5-ml tubes for FACS scanning. Of each with fluorochrome-labeled anti- CD3, CD4, CD8, CD45RA, and CD45RO stained sample, 5000 events were acquired in a forward scatter/sideward mAb as described below. The mean purity of all separations as determined scatter lymphocyte gate. Cell surface marker expression was analyzed on the by FACS staining with mAb specific for CD45RA and CD45RO was CD4ϩ lymphocyte population. Ն95% for each subset. Double-positive (CD45RAϩ/ROϩ) “effector” cells, variably present in the PBMC (9 and our observation), were selected out. Statistical methods Cell viability was always Ͼ98% by trypan blue staining exclusion. The yield of purified cells recovered from all cells loaded into the column Given the difficulty in obtaining large numbers of purified T cells to set up ranged from 25 to 45%, in accordance to the manufacturer’s specifications. full limiting dilution experiments, we chose culture conditions aimed to To assess whether cell loss biased the phenotypic characteristics of T cell obtain MBP-specific T cell lines arising from a single precursor cell per subsets, we performed FACS analysis of cell surface markers including, in well. Such expectation was confirmed by the clonal profiles of TCR usage addition to CD45 isoforms, costimulation receptors (CD28), cell adhesion of the T cell lines (data not shown). This allowed us to estimate T cell molecules (CD11a, CD49d, and CD62L) and activation markers (CD25 precursor frequency of MBP-responding cells by dividing the number of and CD69). The expression of these molecules on the isolated CD45RAϩ responding wells by the number of wells seeded, multiplied by the number or ROϩ subset reflected the expected phenotypes of naive and memory T of cells per well. cells and matched the profiles found on the corresponding subset analyzed Variance was measured by calculating SD. Differences between groups by electronic gating in whole PBMC (data not shown). These experiments were evaluated by the unpaired t test. For data not normally distributed, the also confirmed that the negative selection strategy avoided functional al- Mann-Whitney U rank sum test was applied. Statistical calculations were terations consequent to engagement of CD45 molecules, yielding unaltered performed using SigmaStat software (Jandel Scientific, San Rafael, CA). cells. Results Generation and characterization of Ag-specific T cell clones The human T cell response to MBP originates predominantly T cell clones (TCC) specific for MBP were established from purified T cell from the CD45RAϩCD4ϩ T cell subset subsets using a modification of the split-well method (2). From each donor, purified CD45RAϩ/ROϪ and CD45 RAϪ/ROϩ CD4ϩ T cells (2.5 ϫ 104/ To estimate the frequency of autoantigen-specific T cells in the ϩ well) were seeded into 96-well plates (Nunc, Roskilde, Denmark) along naive and memory resting CD4 T cell population, we obtained AUTOREACTIVE CD4؉ T CELL REPERTOIRE IN NAIVE/MEMORY SUBSETS 5476

Table I. Generation of MBP/peptide-specific TCC from the naive (CD45RAϩ/ROϪ) and resting memory (CD45RAϪ/ROϩ) T cell subsets

MBP-Reactive Wells from Naive T MBP-Reactive Wells from Memory Cell Subsets T Cell Subsets

Subjects No. positive % positive No. positive % positive

MS patients MS1 30/120 25 7/60 12 MS2 24/120 20 0/60 0 MS3 6/30 20 6/42 14 MS4 11/110 10 12/220 5.4 MS5 12/60 20 1/30 3.3 MS6 21/180 11.7 8/90 8.9 MS7 0/30 0 2/120 1.6 Total MS 104/650 16.0 36/622 5.8 HDa HD1 20/90 22 9/90 10 HD2 3/16 18.7 4/60 6.7 HD3 16/120 13 4/120 3 Total HD 39/226 17.2 17/270 6.3 Downloaded from Total all subjects 143/876 16.3 53/892 5.9

a HD, healthy donors. highly purified CD45RAϩ/ROϪ and CD45RAϪ/ROϩ cell subsets inated from the CD45RAϩ subset. T cells responding to other

by negative selection. To assess the maximum proliferative capac- immunodominant regions of MBP, such as MBP(61–79) in the http://www.jimmunol.org/ ity of the isolated subsets in response to potent stimuli, we com- central region of the protein and MBP(141–159) toward the car- pared the primary response to SAg (staphylococcal enteroxin A, boxyl terminus, resided as well in the naive pool (nine naive vs 1 staphylococcal enteroxin B, SEC1–SEC3, staphylococcal en- memory TCC and seven vs none, respectively). Strikingly, when teroxin D, staphylococcal enteroxin E, and toxic shock syndrome considering individual patients, the MBP epitopes recognized by toxin-1) at concentrations ranging from 10Ϫ2 to 102 ng/ml in dose- TCC from either subset excluded each other almost completely response proliferation assays. Cells from both subsets proliferated (Table II). Even in the case of naive- and memory- derived TCC comparably well, indicating that universal T cell stimuli elicit sim- from a given subject responding to the same peptide MBP (111– ilar responses both in naive and memory T lymphocytes. In con- 129) (patient MS4), further epitope mapping with truncated pep- trast, the short-term reaction to TT in primary proliferation assays tides delineated different nested epitopes, probably recognized by by guest on September 26, 2021 clearly resided in the CD45ROϩ subset, as expected from a re- sponse to a recall Ag in immunized subjects. However, although a vigorous primary proliferation to TT (assayed after 3 days) could be detected only in the CD45ROϩ subset, short-term TT-specific cell lines could be raised after rechallenge with the Ag from the CD45RAϩ subset as well (data not shown). We next dissected the T cell response of the subsets to MBP using purified T cell subsets and APCs. Interestingly, it was the CD45RAϩ T cell population that originated predominant re- sponses to MBP, both in MS patients and in healthy controls (Ta- ble I). The estimated precursor frequency of MBP-reactive T cells in the CD45RAϩ subset was significantly higher than in the CD45ROϩ subset of MS patients, (6.10 Ϯ 3.41 ϫ 10Ϫ6 vs 2.59 Ϯ 2.13 ϫ 10Ϫ6; p ϭ 0.040). Although this was true also for healthy donors (7.24 Ϯ 1.79 ϫ 10Ϫ6 vs 2.67 Ϯ 1.33 ϫ 10Ϫ6), the small number of subjects available for analysis does not allow us to compare their precursor frequency of MBP-reactive cells with that of MS patients.

MBP-specific TCC originating from the CD45RAϩ and CD45ROϩ subsets recognize different antigenic epitopes within individual subjects FIGURE 1. Summary of MBP peptide specificities of CD45RAϩ vs To assess the Ag-fine specificities of TCC originating from either ϩ subset, we tested MBP-reactive TCC in proliferation assays using CD45RO subset-originating TCC. The fine Ag specificity of 85 TCC was tested against a set of overlapping peptides spanning the MBP sequence. a panel of 16 overlapping MBP peptides. As summarized in Fig. 1, Included are data from both MS patients and controls. The number of TCC the overall specificities corresponded well to the pattern of immu- specific for each MBP peptide is plotted on the x-axis, distinct in nodominant MBP epitopes previously identified by others and by CD45RAϩ subset-derived (hatched bars) and CD45ROϩ subset-derived us (3–5, 26–28). However, a clear skewing in the specificities of (filled bars). Remarkably, T cell lines specific for the immunodominant TCC derived from either subset emerged. Most important, 13 of 14 epitopes MBP(61–79, 81–99, and 141–159) predominantly emerged from TCC recognizing the immunodominant epitope MBP(81–99) orig- the CD45RAϩCD4ϩ subset. The Journal of Immunology 5477

Table II. Skewed peptide specificity of naive and memory subset-derived MBP-reactive TCC from individual MS patients

Patient MS1 Patient MS3 (DRBI*1501, 04*) (DRBI*0403, 1501) Patient MS4 (DRB1*0401, 1301) Patient MS6 (DRB1*1502, 0701)

TCC Peptide TCC Peptide TCC Peptide TCC Peptide

Naive subset-derived TCC RA2B(8)3 1–19 RA B2 61–79 RA7 81–99 RA1 1–19 RA1A(5)1 81–99 RA B9 61–79 RA2 111–129a RA7 21–39 RA1B(6)4 81–99 RA C6 81–99 RA11 111–129a RA8 41–59 RA1B(6)6 81–99 RA D2 81–99 RA6 131–149 RA10 61–79 RA2B(8)1 141–159 RA D4 81–99 RA4 131–149 RA11 61–79, 81–99 RA2B(8)6 151–170 RA B6 141–159 RA1 141–159 RA2, RA16, 81–99 RA 17 RA2B(8)7 RA 19, RA20, 141–159 RA3 Memory subset-derived TCC RO1A(5)7 11–29 RO B9 MBPb RO2 MBPb RO2 11–29 RO2A(7)3 11–29 RO E4 MBPb RO4 71–89 RO7 11–29 RO1B(10)1 MBPb RO B10 151–170 RO5 31–49 RO8 11–29 RO2B(8)2 71–89 RO C5 151–170 RO1 (11–29), 111–129,c (121–139) RO6(2) 31–49 RO1B(10)3 MBPb RO D4 151–170 RO3 111–129,c (121–139) RO6 101–119 RO F1 151–170 Downloaded from a Epitope MBP(116–124). b Epitope MBP(115–125). In patient MS4, TCC reacting to MBP(111–129) were observed from both the naive and memory subset. However, performing a more detailed analysis of these TCC with a set of truncated peptides revealed the recognition of two distinct minimal epitopes, MBP(116–124) and MBP(115–125), by clones derived from the naive and memory subset, respectively. These clones expressed different TCR (see Results). c Denotes lack of recognition of the overlapping MBP peptide set despite confirmed specificity to the whole protein. http://www.jimmunol.org/ distinct TCC. Consistent with this finding, in this patient naive (L-selectin), and CD27. Cell adhesion molecules under examina- subset-derived MBP(116–124)-specific TCC MS4-RA2 and -RA11 tion included CD54 (ICAM-1), CD11a (LFA-1), CD2 (LFA-2), ␤ and memory-derived MBP(115–125)-specific TCC MS4-RO1, CD58(LFA-3), CD29 ( 1 integrin chain), CD49d (VLA-4), and -RO3 and -RO7 expressed different TCR V␤ genes, i.e., TCRBV3 CD44 (Pgp-1). We also assessed the expression of molecules in- and TCRBV2, respectively. volved in Ag presentation, costimulation, and T cell-B cell inter-

ϩ ϩ actions such as HLA-DR, CD28, CD80 (B7-1), CD86 (B7-2), Cytokine secretion profiles of CD45RA and CD45RO CD152 (CTLA-4), and CD40L. Activation, proliferation and ap- subset-originating TCC optosis markers included CD25 (IL-2R ␣-chain), CD26, CD30,

To define the T helper subtype of MBP-specific T cells emerging CD69, CD134, and CD95 (Fas). Significant differences between by guest on September 26, 2021 from the CD45RAϩ and CD45ROϩ subsets of MS patients, we naive and memory subset-derived TCC are summarized in Fig. 4. measured the secretion of pro (IFN-␥, TNF-␣)- and anti- Interestingly, even after repeated in vitro Ag stimulation of the inflammatory cytokines (IL-4, IL-10) in culture supernatants of 41 TCC, consistent differences in their CD45R isoform expression TCC raised from 4 subjects , as well as the APC-activating factor were still clearly detectable. The proportion of CD28ϩ cells was GM-CSF. The conventional types of T helper cells (Th1, Th2, and greater in memory-derived TCC ( p ϭ 0.003). CD29 expression Th0) emerged from both the naive and the memory subsets (Table was also strongly associated with memory-derived TCC, both in III). By using the IFN-␥ to IL-4 quotient as a marker of Th1- vs terms of percent positive cells ( p Ͻ 0.001) and staining intensity Th2-type cells, we failed to detect clear-cut systematic differences ( p Ͻ 0.001). We observed a more prominent CD27 down-regu- in the cytokine secretion of TCC derived from the naive and mem- lation ( p ϭ 0.018) and a larger proportion of CD134ϩ cells ( p ϭ ory subsets (Fig. 2). Although the small number of memory subset- 0.026) in the memory-derived TCC group. Trends not reaching derived TCC available for analysis precludes a definitive statement statistical significance included a larger number of TCC expressing on their phenotype, naive subset-originating TCC were predomi- CD62Lhigh in the naive subset-derived group and a greater pro- nantly Th1 or Th0, similar to observations on PBMC-derived TCC portion of CD40Lϩ cells in the memory-derived TCC (data not specific for immunodominant MBP epitopes (5, 29). shown). The percentages of CD2-, CD11a-, CD49d-, CD58-,

ϩ CD26-, and CD44-positive cells neared 100% in almost all TCC, Higher functional avidity of epitope recognition by CD45RA with comparable fluorescence intensities. Similarly, Ͼ95% of all subset-derived TCC does not depend on higher expression of TCC tested were CD54ϩ. As expected from in vitro-stimulated T costimulatory/accessory molecules cells, both naive and memory TCC included Ͼ80% of cells with To estimate the functional avidity for the Ag of naive vs memory significant HLA-DR surface expression. Although there were only Ͻ ϩ subset-derived MBP-specific T cells, we determined the EC50 of few ( 10%) CD152 cells in each TCC, variable proportions of 35 representative, well-characterized TCC (25 naive and 10 mem- CD25-, CD30-, CD80-, and CD86-positive cells were observed, ory derived). CD45RAϩ subset-derived TCC had significantly however, without systematic differences. Finally, there were no ϩ lower EC50 than CD45RO subset-derived TCC (0.50 vs 6.25 significant differences in the surface expression of the apoptosis- ␮ Ͻ g/ml, median; p 0.001, Fig. 3). Since the EC50 measure does mediating molecule CD95 (Fas) on TCC derived from the not represent the receptor/ligand affinity in physicochemical terms, CD45RAϩ or CD45ROϩ subsets. but rather a functional equivalent of complex interactions involv- This comprehensive analysis demonstrated that the lower Ag ing a number of molecules, we examined the expression of the concentration required by CD45RA subset-derived TCC was not most important cofactors involved in cell proliferation in these secondary to a more favorable expression of molecules for acces- TCC. Among molecules regulating signaling and homing of naive sory interactions, in particular ligands for costimulation or adhe- and memory T cells, we analyzed CD45RA, CD45RO, CD62L sion molecules. Although it should be pointed out that recognition AUTOREACTIVE CD4؉ T CELL REPERTOIRE IN NAIVE/MEMORY SUBSETS 5478

Table III. Cytokine secretion of naive and memory subset-derived TCCa

TCC IFN-␥* TNF-␣ IL-4 GM-CSF IL-10

MS3-RAB2 165.6 307.2 222.8 710.7 20.7 MS3-RAB9 38.5 315.7 96.7 212.6 0 MS3-RAC6 56.5 131.1 260.2 390.5 0 MS3-RAD4 0 0 14.9 40.9 0 MS3-ROC5 0.8 125 80.2 76.2 3.1 MS3-ROD4 0 0 985.3 649.5 42.7 MS4-RA1 571.1 2264.9 23.9 2231.8 130.6 MS4-RA11 83.2 683.8 88.2 757.4 44.1 MS4-RA2 32.1 356.7 46.6 43.6 1.2 MS4-RA4 63.9 416.9 27.9 59 11.1 MS4-RA7 30.9 0 0 1553.3 4.7 MS4-RO1 129.9 807.9 0 253.6 0 MS4-RO2 74.8 179.2 216.7 120.3 29.6 MS4-RO3 21.8 0 37 27.8 26.7 MS4-RO4 436.5 738.7 0 467.3 0 ϩ MS4-RO5 175.5 376.4 100.9 388.7 77.7 FIGURE 2. T helper phenotype of TCC originating from CD45RA and CD45ROϩ subsets of MS patients. The IFN-␥ vs IL-4 secretion of 41 Downloaded from MS5-RA1 6221.5 1913 36.9 2596.5 17.8 TCC stimulated with 10 ␮g/ml specific antigenic peptide delineates Th0, MS5-RA12 4619.5 1328.4 18.5 3037.4 0 ϩ Th1, and Th2 subtypes. CD45RA subset-derived TCC are represented by MS5-RA2 5984.5 2281.4 17.5 2347.5 0 ϩ MS5-RA3 6286.6 2658 20.7 2724.1 35.4 open circles and CD45RO subset-derived TCC by cross-hatched circles (see MS5-RA4 6406.1 2339.3 20.5 2527.9 56.4 Results). Overlapping of some circles corresponding to TCC with nearly iden- MS5-RA5 3680 836.1 33.8 1628 22.2 tical cytokine secretion may prevent their individual visualization. MS5-RA6 1293.4 1039.6 0 625.6 0

MS5-RA9 1441.4 685.7 20.7 742.9 0 http://www.jimmunol.org/ MS5-RO1 2985.4 1833.8 25.8 716 23.4 MS6-RA1 678 1441.4 79.5 1411.8 60.4 were lower in the purified CD45RAϩ subset, thus penalizing rather MS6-RA10 1401.1 1983.1 29 2453.7 2.5 than favoring its proliferation potential (11). Our results are in MS6-RA11 1305.3 2677.6 43.3 2275.6 0 contrast to recent data reporting a higher frequency of MBP-spe- MS6-RA14 42.5 185.5 19.1 59 0 ϩ MS6-RA16 17.5 177.2 19.2 99.4 0 cific T cells in memory CD45RO subsets from MS patients (30). MS6-RA17 707.8 2364.3 53.6 1553.3 29.4 The different experimental setting might account for this discrep- MS6-RA19 28.5 1274.5 79.5 757.4 78.8 ancy. Burns et al. (30) generated MBP-specific T cell lines from MS6-RA20 30.1 1045.9 163.3 294.1 0 seeding PBMC populations depleted of cells expressing CD45RA MS6-RA21 536.6 2888.9 54.3 1368.9 0 by guest on September 26, 2021 MS6-RA3 898.8 952.8 19.1 781.9 117.3 or CD45RO. Since not only lymphocytes, but also dendritic cells MS6-RA5 1979.8 2545 51.8 1380 10 and monocytes express CD45R isoforms (Refs. 3 and 32 and our MS6-RA6 5094.3 2929.9 31.1 2084.4 56.8 observation), isoform depletion may affect Ag presentation by ac- MS6-RA7 625.9 1672.1 22.5 792 0 cessory cells, which is crucial for the activation of naive T lym- MS6-RA8 4263.2 3813.6 22.1 4047.2 131.1 MS6-RO6 658.4 2889.9 0 1512.7 117 phocytes (6, 11, 33). Different from that study, we compared the MS6-RO7 207.3 947.6 0 1021.1 0 precursor frequency in the two subsets using exact numbers of isolated CD45RAϩ and CD45ROϩ T cells and delivering anti- a For all cytokines, values refer to 48-h supernatants obtained after stimulation with 10 ␮g/ml MBP peptide. Background values were subtracted from those of stim- genic presentation by means of unmanipulated PBMC. ulated cultures. The second major finding of our study is that the human T cell response to immunodominant MBP epitopes mainly resides in the CD45RAϩ/ROϪ naive subset. This observation is in line with re- of epitopes with higher binding affinity for the MHC complex such sults obtained in experimental models of autoimmunity. Enceph- as MBP(81–99) by naive-derived cells might contribute to their alitogenic T cells with specificity for the immunodominant regions lower EC values, our data show that T cells responding to MBP 50 of MBP could be selected in vitro from PBL and spleen cells of with overall greater efficiency are selected from the CD45RAϩ nonimmunized healthy Lewis rats (34). Similarly, the T cell rep- repertoire. ertoire of naive healthy rats harbored diabetogenic T cells (35). In addition, CD45RB high cells have autoaggressive potential toward Discussion several other target organs (36, 37). Unlike the case in inbred an- In this study, we have dissected the response of highly purified, imal strains, however, MBP-reactive cells from either the naive or putative naive and memory-resting CD45RAϩ/ROϪ and memory CD4ϩ T subsets of MS patients did not show the strong CD45RAϪ/ROϩ CD4ϩ T cell subsets against the human self-Ag bias toward a Th1 phenotype which is characteristic for disease- MBP. Our findings suggest a major role of the naive T cell subset mediating effector cells in experimental models of autoimmune in the origin and the maintenance of the self-reactive effector diseases, but were phenotypically more diverse. CD4ϩ peripheral T cell repertoire. Although a prevalent T cell reactivity to MBP from the naive This notion is first supported by the ϳ2-fold higher frequency of subset in MS patients could also be seen as a lack of involvement MBP-reactive T cells in the naive CD45RAϩ/ROϪ subset that did of this autoantigen in the pathogenesis of the disease, the detection not depend on a biased viability, proliferative potential, or core- of proinflammatory and activated MBP-specific T cells in MS pa- ceptor/adhesion molecule density of the progenitor T cell popula- tients by different functional assays argues against this possibility tion. In fact, viability and response to potent stimuli like SAg did (38–40). Moreover, the increased frequency of MBP-specific T not differ in the naive and memory cell subsets. Levels of expres- cells which can be raised in the absence of CD28/B7-mediated sion of IL-2R, adhesion molecules, and ligands for costimulation costimulation in MS patients (41, 42) has suggested the presence The Journal of Immunology 5479 Downloaded from ϩ ϩ ϭ FIGURE 3. Functional antigenic avidity of CD45RA and CD45RO subset-derived TCC. A, Summarizes the analysis of the EC50 of naive (n 25) and memory (n ϭ 10) subset-derived TCC, which revealed a significantly more efficient recognition of antigenic epitopes by naive-originating T cells. Central tendency measure is the median. Boxes frame the 5th to the 95th percentiles, and error bars represent SD. Representative examples of dose-response proliferation curves of a naive-derived TCC (B, TCC MS6-RA17) and a memory-derived TCC (C, TCC MS6-RO6) are given.

of myelin-reactive activated/memory cells. However, MBP-spe- nance of the autoreactive effector T cell pool. In this context, par- http://www.jimmunol.org/ cific responses in those studies were not examined with respect to ticularly significant are the more efficient recognition of antigenic their origin in the CD45RAϩ vs CD45ROϩ compartments. The epitopes by naive subset-derived TCC and the absence, in each CD45RAϩ T cell response to recall Ag increases 3- to 5-fold in the individual subject, of resting memory T cells responding to epitopes presence of anti-CD28 Ab (43), but it remains to be determined to recognized by the naive repertoire. Given the lower density of core- which extent this population contributes to the overall response of ceptors like CD28 and CD29 observed on naive subset-derived cells, PBMC in the absence of costimulation. their Ag-specific responses at 10-fold lower concentrations appear to The differences we found in the human CD45RAϩ and be significant and indicate that a greater functional avidity of TCR- CD45ROϩ MBP-specific T cell repertoires offer important clues to MHC-peptide interactions outweighed poorer costimulation. The speculate on the possible mechanisms of regulation and mainte- absence within each individual of memory T cells responding to by guest on September 26, 2021

FIGURE 4. Differential expression of cell sur- face molecules on naive vs memory subset-derived TCC. An extensive mAb panel was used to compare cell surface marker expression on 20 naive and 15 memory subset-derived TCC (see Materials and Methods). Only statistically significant differences are shown. The upper plots show CD45 isoform- expressing cell percentages (left panel) and mean fluorescence intensity (MFI, center panel) for naive- derived (open circles) and memory-derived (cross- hatched circles) TCC. A relatively decreased CD27 expression was detectable in memory subset-de- rived T cells as compared with the naive derived (upper right panel). The lower plots show the sig- nificantly different proportions (% positive) and in- tensity of expression (MFI) of CD28 (left panel), CD29 (center panel), and CD134 (right panel). Vi- sualization of all TCC may be hampered in some cases by the overlapping of circles corresponding to very close values. Asterisks at p values indicate the use of the Mann-Whitney U rank sum test. AUTOREACTIVE CD4؉ T CELL REPERTOIRE IN NAIVE/MEMORY SUBSETS 5480 epitopes recognized by the naive repertoire is difficult to reconcile 4. Ota, K., M. Matsui, E. L. Milford, G. A. Mackin, H. L. Weiner, and D. A. Hafler. with the hypothesis that primed MBP-specific cells might revert to 1990. T-cell recognition of an immunodominant myelin basic protein epitope in multiple sclerosis. Nature 346:183. a canonical naive phenotype, as shown in other systems (44). 5. Muraro, P. A., M. Vergelli, M. Kalbus, D. Banks, J. W. Nagle, L. R. Tranquill, Rather, the lack of MBP epitopes shared by the naive and memory G. T. Nepom, W. E. Biddison, H. F. McFarland, and R. Martin. 1997. Immu- nodominance of a low-affinity MHC binding myelin basic protein epitope (res- subsets in MS patients suggests that naive T cells specific for the idues 111–129) in HLA-DR4 (B1*0401) subjects is associated with a restricted immunodominant regions of MBP do not evolve beyond effector T cell receptor repertoire. J. Clin. Invest. 100:339. stage into resting long-term memory cells with CD45RAϪ/ROϩ 6. Matzinger, P. 1994. Tolerance, danger, and the extended family. Annu. Rev. phenotype after priming and activation in vivo. Immunol. 12:991–1045:991. 7. Sprent, J. 1994. T and B memory cells. Cell 76:315. We propose that in MS, upon effective Ag priming probably due 8. Swain, S. L., M. Croft, C. Dubey, L. Haynes, P. Rogers, X. Zhang, and to a potent cross-reacting stimulus (45), sufficient TCR affinity, L. M. Bradley. 1996. From naive to memory T cells. Immunol. Rev. 150:143. and adequate costimulation, naive CD45RAϩ T cells become ac- 9. Byrne, J. A., J. L. Butler, and M. D. Cooper. 1988. Differential activation re- quirements for virgin and memory T cells. J. Immunol. 141:3249. tivated and proliferate in secondary lymphoid organs, acquiring a 10. Croft, M., and S. L. Swain. 1995. Recently activated naive CD4 T cells can help ϩ ϩ double-positive (CD45RA /RO ) effector cell phenotype. These resting B cells, and can produce sufficient autocrine IL-4 to drive differentiation activated cells can reach the brain vascular endothelium via the to secretion of T helper 2-type cytokines. J. Immunol. 154:4269. 11. Dubey, C., M. Croft, and S. L. Swain. 1995. Costimulatory requirements of naive peripheral blood and cross the blood-brain barrier. Their recogni- CD4ϩ T cells. ICAM-1 or B7-1 can costimulate naive CD4 T cell activation but tion of immunodominant epitopes of myelin Ag may elicit proin- both are required for optimum response. J. Immunol. 155:45. flammatory T cell functions and initiate a demyelinating attack. To 12. Janeway, C. A. J. 1992. The T cell receptor as a multicomponent signalling machine: CD4/CD8 coreceptors and CD45 in T cell activation. Annu. Rev. Im- limit the autoimmune process and restore self-tolerance, high-af- munol. 10:645.

finity MBP-primed T cells need to be neutralized by peripheral 13. Lee, W. T., and E. S. Vitetta. 1991. The differential expression of homing and Downloaded from mechanisms, including clonal deletion and high-dose anergy (46, adhesion molecules on virgin and memory T cells in the mouse. Cell. Immunol. 132:215. 47). This concept is supported by the recent observation of a strong 14. Schweighoffer, T., G. E. Luce, Y. Tanaka, and S. Shaw. 1994. Differential ex- T cell response to an immunodominant epitope in MBP-deficient pression of integrins ␣6 and ␣4 determines pathways in human peripheral CD4ϩ (shiverer) mice, and the inactivation of the high-avidity MBP-spe- T cell differentiation. Cell Adhes. Commun. 2:403. 15. Horgan, K. J., G. E. Luce, Y. Tanaka, T. Schweighoffer, Y. Shimizu, cific repertoire in congenic MBP-expressing (C3H) mice (48). Ac- S. O. Sharrow, and S. Shaw. 1992. Differential expression of VLA-␣4 and ϩ ␤ ϩ ϩ cording to a recently proposed model (23), memory CD45RO VLA- 1 discriminates multiple subsets of CD4 CD45R0 “memory” T cells. http://www.jimmunol.org/ cells, which we found to recognize subdominant MBP epitopes J. Immunol. 149:4082. 16. Bradley, L. M., S. R. Watson, and S. L. Swain. 1994. Entry of naive CD4 T cells with low affinity, may have differentiated directly from the naive into peripheral lymph nodes requires L-selectin. J. Exp. Med. 180:2401. pool as a result of partial activation, bypassing the proliferative/ 17. Smith, S. H., M. H. Brown, D. Rowe, R. E. Callard, and P. C. L. Beverley. 1986. effector stage. These cells could survive in the peripheral blood Functional subsets of human help-inducer cells defined by a new monoclonal antibody, UCHL1. Immunology 58:63. as a result of cross-reactive stimulation, facilitated by their high 18. Akbar, A. N., L. Terry, A. Timms, P. C. L. Beverley, and G. Janossy. 1988. Loss density of adhesion molecules and receptors for costimulation of CD45R and gain of UCHL1 reactivity is a feature of primed T cells. J. Im- (46, 49). munol. 140:2171. ϩ 19. Vitetta, E. S., M. T. Berton, C. Burger, M. Kepron, W. T. Lee, and X. M. Yin. Our interpretation implies that effector CD4 cells mediating 1991. Memory B and T cells. Annu. Rev. Immunol. 9:193.

ϩ by guest on September 26, 2021 demyelination in MS are generated de novo from the CD45RA T 20. Bell, E. B., and S. M. Sparshott. 1990. Interconversion of CD45R subsets of CD4 cell pool and differentiate either into short-lived effector or long- T cells in vivo. Nature 348:163. 21. Bunce, C., and E. B. Bell. 1997. CD45RC isoforms define two types of CD4 term memory cells. Defining how naive cells can be recruited into memory T cells, one of which depends on persisting antigen. J. Exp. Med. 185: an inflammatory focus leading to organ-specific immune attack 767. warrants further studies. The detection of prevalent, immunodom- 22. Michie, C. A., A. R. McLean, C. Alcock, and P. C. L. Beverley. 1992. Lifespan inant autoantigen-specific responses from the CD45RAϩ/ROϪ of human lymphocyte subsets defined by CD45 isoforms. Nature 360:264. ϩ 23. Farber, D. 1998. Differential TCR signaling and the generation of memory cells. CD4 T cell compartment points to this subset as the main reser- J. Immunol. 160:535. voir of self-reactive cells, continuously available to trigger human 24. Deibler, G. E., R. E. Martenson, and M. W. Kies. 1972. Large scale preparation of myelin basic protein from central nervous tissue of several mammalian spe- autoimmune processes upon the appropriate stimuli. cies. Prep. Biochem. 2:139. 25. Rothstein, D. M., A. Yamada, S. F. Schlossman, and C. Morimoto. 1991. Cyclic regulation of CD45 isoform expression in a long term human CD4ϩ CD45RAϩ Acknowledgments T cell line. J. Immunol. 146:1175. We thank D. F. Pette for her expert technical assistance, Dr. S. Leitman 26. Chou, Y. K., P. Henderikx, M. Vainiene, R. H. Whitham, D. N. Bourdette, C. H. Chou, G. A. Hashim, H. Offner, and A. A. Vandenbark. 1991. Specificity (Department of Transfusion Medicine, National Institutes of Health) for of human T cell clones reactive to immunodominant epitopes of myelin basic coordinating leukaphereses, and T. Simonis (HLA Laboratory, National protein. J. Neurosci. Res. 28:280. Institutes of Health) for HLA typing of the subjects included in this study. 27. Zhang, J., R. Medaer, G. A. Hashim, Y. Chin, E. van den Berg-Loonen, and We are also thankful to Prof. W. Sebald (Institut fu¨r Physiologische Che- J. C. Raus. 1992. Myelin basic protein-specific T lymphocytes in multiple scle- rosis and controls: precursor frequency, fine specificity, and cytotoxicity. Ann. mie II, University of Wu¨rzburg, Wu¨rzburg, Germany) for providing rIL-2, Neurol. 32:330. Drs. N. Ling (Neurocrine, San Diego, CA), T. Spack (formerly at Anergen, 28. Valli, A., A. Sette, L. Kappos, C. Oseroff, J. Sidney, G. Miescher, Redwood City, CA), and R. Houghten (Multiple Peptide Systems, San M. Hochberger, E. D. Albert, and L. Adorini. 1993. Binding of myelin basic Diego, CA) for providing MBP peptides, and Dr. S. Stu¨rzebecher for crit- protein peptides to human histocompatibility leukocyte antigen class II molecules ically reading this manuscript. and their recognition by T cells from multiple sclerosis patients. J. Clin. Invest. 91:616. 29. Hemmer, B., M. Vergelli, P. A. Calabresi, T. Huang, H. F. McFarland, and References R. Martin. 1996. Cytokine phenotype of human autoreactive T cell clones specific for the immunodominant myelin basic protein peptide (83–99). J. Neurosci. Res. 1. Martin, R., H. F. McFarland, and D. E. McFarlin. 1992. Immunological aspects 45:852. of demyelinating diseases. Annu. Rev. Immunol. 10:153. 30. Burns, J., B. Bartholomew, and S. Lobo. 1999. Isolation of MBP-specific T cells 2. Pette, M., K. Fujita, B. Kitze, J. N. Whitaker, E. Albert, L. Kappos, and predominantly from the memory T cell compartment in multiple sclerosis. Ann. H. Wekerle. 1990. Myelin basic protein-specific T lymphocyte lines from MS Neurol. 45:33. patients and healthy individuals. Neurology 40:1770. 31. Prickett, T. C., and D. N. Hart. 1990. Anti-leukocyte common (CD45) antibodies 3. Martin, R., D. Jaraquemada, M. Flerlage, J. R. Richert, J. N. Whitaker, inhibit dendritic cell stimulation of CD4 and CD8 T-lymphocyte proliferation. E. O. Long, D. E. McFarlin, H. F. McFarland, J. Richert, and J. Whitaker. 1990. Immunology 69:250. Fine specificity and HLA restriction of myelin basic protein-specific cytotoxic T 32. Wood, G. S., P. S. Freudenthal, A. Edinger, R. M. Steinman, and R. A. Warnke. cell lines from multiple sclerosis patients and healthy individuals. J. Immunol. 1991. CD45 epitope mapping of human CD1aϩ dendritic cells and peripheral 145:540. blood dendritic cells. Am. J. Pathol. 138:1451. The Journal of Immunology 5481

33. Croft, M., L. M. Bradley, and S. L. Swain. 1994. Naive versus memory CD4 T basic protein and proteolipid protein in peripheral blood and cerebrospinal fluid cell response to antigen: memory cells are less dependent on accessory cell co- of patients with multiple sclerosis. J. Exp. Med. 179:973. stimulation and can respond to many antigen-presenting cell types including rest- 41. Scholz, C., K. T. Patton, D. E. Anderson, G. J. Freeman, and D. A. Hafler. 1998. ing B cells. J. Immunol. 152:2675. Expansion of autoreactive T cells in multiple sclerosis is independent of exoge- 34. Schluesener, H. J., and H. Wekerle. 1985. Autoaggressive T lymphocyte lines nous B7 costimulation. J. Immunol. 160:1523. recognizing the encephalitogenic region of myelin basic protein: in vitro selection 42. Lovett-Racke, A. E., J. L. Trotter, J. Lauber, P. J. Perrin, C. H. June, and from unprimed rat T lymphocyte populations. J. Immunol. 135:3128. M. K. Racke. 1998. Decreased dependence of myelin basic protein-reactive T 35. Fowell, D., and D. Mason. 1993. Evidence that the T cell repertoire of normal rats cells on CD28-mediated costimulation in multiple sclerosis patients. J. Clin. In- contains cells with the potential to cause diabetes: characterization of the CD4ϩ vest. 101:725. T cell subset that inhibits this autoimmune potential. J. Exp. Med. 177:627. 43. Pilling, D., A. N. Akbar, P. A. Bacon, and M. Salmon. 1996. CD4ϩ CD45RAϩ 36. Powrie, F., and D. Mason. 1990. OX-22high CD4ϩ T cells induce wasting disease T cells from adults respond to recall antigens after CD28 ligation. Int. Immunol. with multiple organ pathology: prevention by the OX-22low subset. J. Exp. Med. 11:1737. 172:1701. 44. Bell, E. B. 1998. CD4ϩ T-cell memory, CD45R subsets and the persistence of 37. Powrie, F., R. Correa-Oliveira, S. Mauze, and R. Coffman. 1994. Regulatory antigen: a unifying concept. Immunol. Today 19:60. interactions between CD45RBhigh and CD45RBlow CD4ϩ T cells are important 45. Hemmer, B., B. T. Fleckenstein, M. Vergelli, G. Jung, H. F. McFarland, for the balance between protective and pathogenic cell-mediated immunity. R. Martin, and K.-H. Wiesmuller. 1997. Identification of high potency microbial J. Exp. Med. 179:589. and self ligands for a human autoreactive class II-restricted T cell clone. J. Exp. 38. Olsson, T., W. W. Zhi, B. Hojeberg, V. Kostulas, Y. P. Jiang, G. Anderson, Med. 185:1651. H. P. Ekre, and H. Link. 1990. Autoreactive T lymphocytes in multiple 46. Mason, D. 1998. A very high level of crossreactivity is an essential feature of the sclerosis determined by antigen-induced secretion of interferon-␥. J. Clin. T-cell receptor. Immunol. Today 19:395. Invest. 86:981. 47. Hayashi, R. J., D. Y. Loh, O. Kanagawa, and F. Wang. 1998. Differences between 39. Allegretta, M., J. A. Nicklas, S. Sriram, and R. J. Albertini. 1990. T cells re- responses of naive and activated T cells to anergy induction. J. Immunol. 160:33. sponsive to myelin basic protein in patients with multiple sclerosis. Science 247: 48. Targoni, O. S., and P. V. Lehmann. 1998. Endogenous myelin basic protein 718. inactivates the high avidity T cell repertoire. J. Exp. Med. 187:2055. 40. Zhang, J., S. Markovic-Plese, B. Lacet, J. Raus, H. L. Weiner, and D. A. Hafler. 49. Beverley, P. C. 1990. Is T-cell memory maintained by crossreactive stimulation? 1994. Increased frequency of interleukin 2-responsive T cells specific for myelin Immunol. Today 11:203. Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021